Abstract This proposal addresses basic mechanisms by which DNA replication origin function maintains genome stability and the contribution of deficient replication origin licensing to the genomic changes that drive cancer. During origin licensing, origin recognition complex (ORC), Cdc6 and Cdt1 load an inactive form of the minichromosome maintenance protein (MCM) 2-7 hexameric helicase onto chromatin to form pre-replication complexes (pre-RC). At S-phase the helicase activity of MCM2-7 is activated to initiate replication. MCM2-7 is loaded onto chromatin in excess where the excess complexes are dormant unless required to reinitiate replication in the event of stalling of adjacent active replication forks. Using a transgenic mouse model in which MCM2 levels are reduced to ~1/3 of normal, we have shown that insufficient DNA replication origin licensing is a potent driver of replication related errors, predominately focal deletions, leading to loss of tumor suppressor genes and cancer. Using a novel method of short nascent strand analysis to map replication origins we have shown that limiting concentrations of MCM2-7 leads to preferential loss of licensing at discrete genomic locations. A subset of these locations correlates with sites of recurrent focal deletions in tumors that arise in these mice and affect tumor suppressor genes. Further, genetic background affects locations where origin function is lost in parallel with the locations where focal deletions occur and correlate with tumor incidence in MCM2 deficient mice. Here we will directly test the relationship between replication origin function, genomic damage, and cancer incidence by disrupting an origin adjacent to a tumor suppressor gene. Specifically, in aim 1 we will refine a methodology for measuring under-replicated regions of the genome and characterize these regions genome wide in wt and MCM2 deficient mice. In aim 2 we will utilize this method as a readout for under-replication to assay a systematic deletion series across a 60 kbp domain in wt and MCM2 deficient MEFs and map sequences required for origin function. The relationship between functional elements and sites identified by SNS analysis as active origins will be established. The 60 kbp domain contains a tumor suppressor gene and once elements required for origin function are defined, we will target these elements using CRISPR-Cas9 to generate a mouse model in which origin function at this location is lost. These mice will be crossed with MCM2 deficient mice and the consequences of loss of origin function on deletion of the tumor suppressor gene and tumor incidence will be determined in wt mice in comparison to MCM2 deficient mice in which the ability of cells to compensate for loss of the origin by activation of dormant origins is limited. In aim 3 we will examine the interaction between loss of origin function and replication stress induced by chemotherapeutic agents (HU and gemcitabine) in determining sites of chromosome fragility. The effects of modulating MCM2-7 levels using CDK inhibitors on HU and gemcitabine sensitive sites will be determined.